Neuregulin-ErbB Signaling in Neuronal Development and Psychiatric Disorders

1. 单个跨膜神经调节蛋白（TM）在中枢神经元中的亚细胞分布和功能：通过使用四种不同的基因（NRG1- nrg4）、启动子（NRG1: i型、-II型和-II型）和选择性剪接产生大量的神经调节蛋白（NRGs），但这种进化保守多样性的功能意义仍然知之甚少。在发育和成熟的大脑中，促进NRG配体从无活性的前形态转化为能够结合ErbB4受体介导其上述生物学效应的信号胜任配体的细胞和分子过程大部分仍不清楚。正如本文和下文所讨论的，我们最近的研究表明，NRGs可以根据其不同的膜拓扑进行分类，这些膜拓扑赋予了根本不同的亚细胞运输特性。NRGs的前形态包含一个单一的跨膜结构域，如NRG1 （I型和II型）和NRG2，其靶向细胞体质膜，并在与底层内质网的专门接触部位积累。首先，我们分析了NRG2，一个在发育中的出生后和成人中枢神经系统中显著表达的同型。利用新的双标记原位杂交技术（RNAScope）和新生成的单克隆抗体，我们发现在啮齿动物海马中，NRG2 mRNA和蛋白在ErbB4阳性GABAergic中间神经元中高表达，表明NRG2可以参与自分泌ErbB4信号传导。有趣的是，我们在轴突中没有发现NRG2蛋白的证据；相反，我们发现未加工的proNRG2在gaba能中间神经元质膜上的大量体树突点积累（Vullhorst et al., Nat comm1;6:7222, 2015）。我们最近对其他单一TM NRGs （NRG1 I型和II型）的研究显示了类似的亚细胞分布。此外，我们发现单个TM的胞外结构域被脱落酶以活性依赖的方式切割，以旁分泌和自分泌方式发出信号，因为皮层中间神经元上的NMDA受体激活促进proNRG2脱落，进而激活ErbB4受体信号传导。ErbB4的激活促进了其与NMDARs的关联及其内化。在这种方式下，NRG/ErbB4和NMDAR之间存在双向信号通路，可以作为调节神经元间兴奋性的稳态机制。

1. Subcellular Distribution and Functions of Single Transmembrane (TM) Neuregulins in Central Neurons: Numerous Neuregulins (NRGs) are generated through the use of four different genes (NRG1-NRG4), promoters (NRG1: types -I, -II and -II)) and alternative splicing, but the functional significance of this evolutionary conserved diversity remains poorly understood. The cellular and molecular processes that promote the conversion of NRG ligands from inactive pro-forms to signaling-competent ligands that can engage ErbB4 receptors to mediate their aforementioned biological effects in the developing and maturing brain remain mostly unknown. As discussed here and below, our recent studies reveal that NRGs can be categorized by their distinct membrane topologies that impart fundamentally different subcellular trafficking properties. NRGs whose pro-forms contain a single transmembrane domain, like NRG1 (types I and II) and NRG2, target to the cell body plasma membrane where they accumulate at specialized contact sites with the underlying endoplasmic reticulum. First we analyzed NRG2, an isotype that is prominently expressed in the developing postnatal and adult CNS. Using a novel double-labeling in situ hybridization technique (RNAScope) and newly generated monoclonal antibodies, we found that in the rodent hippocampus NRG2 mRNA and protein are highly expressed in ErbB4-positive GABAergic interneurons, suggesting that NRG2 can engage in autocrine ErbB4 signaling. Interestingly, we found no evidence of NRG2 protein in axons; instead, we found that unprocessed proNRG2 accumulates at large somato-dendritic puncta on the plasma membrane of GABAergic interneurons (Vullhorst et al., Nat Commun 1;6:7222, 2015). Our more recent studies on the other single TM NRGs (NRG1 types I and II) demonstrate a similar subcellular distribution. Moreover, we found that the ectodomains of single TM are cleaved by sheddases in an activity-dependent manner to signal in paracrine and autocrine fashion, as NMDA receptor activation on cortical interneurons promotes proNRG2 shedding that in turn activates ErbB4 receptor signaling. The activation of ErbB4 promotes its association with NMDARs and their internalization. In this fashion, there is a bidirectional signaling pathway between NRG/ErbB4 and NMDAR that can function as a homeostatic mechanism to regulate interneuron excitability. 2. Subcellular Distribution of Dual Transmembrane (TM) Neuregulins in Central Neurons: By contrast single TM NRGs, we found that dual TM NRGs, such as CRD type III-NRG1 and NRG3 (our recent studies uncovered it also is a dual transmembrane protein) are targeted to axons where they signal in juxtacrine mode. These findings reveal a previously unknown functional relationship between membrane topology and subcellular targeting, and suggest that single- and dual-pass NRGs regulate neuronal functions in fundamentally different ways. This work was supported by a Directors Investigator Award and has recently been submitted for publication (Vullhorst et al., Journal of Neuroscience 37(21):5232-5249). 3. Neuregulin-2 Knockout Mice Exhibit Dopamine Dysregulation and Severe Behavioral Phenotypes with Relevance to Psychiatric Disorders: We found that NRG2 expression in the adult rodent brain does not overlap with NRG1 and is more extensive than originally reported, including expression in the striatum and medial prefrontal cortex (mPFC), and therefore generated NRG2 knockout mice (KO) to study its function. NRG2 KOs have higher extracellular dopamine levels in the dorsal striatum but lower levels in the mPFC; a pattern with similarities to dopamine dysbalance in schizophrenia. Like ErbB4 KO mice, NRG2 KOs performed abnormally in a battery of behavioral tasks relevant to psychiatric disorders. NRG2 KOs exhibit hyperactivity in a novelty-induced open field, deficits in prepulse inhibition, hypersensitivity to amphetamine, antisocial behaviors, reduced anxiety-like behavior in the elevated plus maze and deficits in the T-maze alteration reward test-a task dependent on hippocampal and mPFC function. Acute administration of clozapine rapidly increased extracellular dopamine levels in the mPFC and improved alternation T-maze performance. Similar to mice treated chronically with N-methyl-d-aspartate receptor (NMDAR) antagonists, we demonstrate that NMDAR synaptic currents in NRG2 KOs are augmented at hippocampal glutamatergic synapses and are more sensitive to ifenprodil, indicating an increased contribution of GluN2B-containing NMDARs. Our findings reveal a novel role for NRG2 in the modulation of behaviors with relevance to psychiatric disorders (Yan et al. Molecular Psychiatry 2017). 4. Analysis of ErbB4 function in mice harboring targeted mutations in GABAergic and dopaminergic neurons: Dysfunctional NRG-ErbB4 signaling in the hippocampus, pre-frontal cortex (PFC) and striatum may contribute to alterations in dopamine (DA) function associated with several schizophrenia symptoms. Because we have shown that NRG1 acutely increases extracellular DA levels to regulate LTP and gamma oscillations, and that ErbB4 receptor expression is confined to GABAergic interneurons (cortex) and TH+ mesocortical DAergic neurons, we have used genetic, biochemical and behavioral approaches to measure DA function in the hippocampus, PFC and striatum in mice harboring targeted mutations of ErbB4 in either PV+ or TH+ neurons. Interestingly, we have found that in contrast to GABAergic neurons, ErbB4 is highly expressed on the axons of DA neurons, suggesting that NRG/ErbB4 signaling may directly regulate the presynaptic function of these neurons. We found NRG regulates the increase in extracellular DA levels, at least in part, by regulating DAT function. In contrast to mice harboring CNS-wide or GABAergic-restricted mutation of ErbB4, which show sensory-motor gating deficits and increases in motor activity, mice with ablation of ErbB4 in TH+ neurons only manifest behavioral deficits in cognitive-related tasks, such as: performance on the T-maze, Y-maze and Barnes maze. Our findings suggest that direct effects of NRG/ErbB4 signaling in GABAergic and DAergic neurons in combination regulate cortical circuits and DA homeostasis to affect numerous behaviors relevant to schizophrenia (Skirzewski et al., Molecular Psychiatry 2017). 5. Effects of ketamine on cortical gamma oscillations and role of dopamine receptors. Mounting evidence suggests that gamma oscillations are atypically high at baseline in disorders that affect attention such as schizophrenia and ADHD. Ketamine, an antagonist of the NMDAR that elicits psychosis and affects cognitive functions in healthy individuals that phenocopy schizophrenia. In collaboration with Dr. Judith Walters lab, we are using multi-electrode recordings from the medial prefrontal cortex and dorsomedial thalamus of rats acutely treated with ketamine to analyze the effects of D4 and ErbB4targeting drugs on gamma oscillations in this rodent model with "face validity" for schizophrenia (Furth et al. Psychopharmacology, in review).